The search for agonists and antagonists of cellular receptors has been an intense area of research aimed at drug discovery due to the elegant specificity of these molecular targets. Being able to generate activating mutations in a receptor can be useful in many different ways. For instance, the superfamily of G-protein coupled receptors (GPCRs) represents one of the most important families of drug targets for the pharmaceutical industry. They are activated by a wide range of extracellular signals including small biogenic amines, large protein hormones, neuropeptides, chemokines, lipid-derived mediators and even proteases such as thrombin. They are also fundamental receptors for the sensory perception of light, taste and smell. Moreover, of the top 200 best selling prescription drugs, more than 20% interact with GPCRs, providing worldwide sales of over $20 billion.
GPCRs transduce their signals across the plasma membrane via an interaction with heterotrimeric G-proteins, and this leads to an activation of intracellular effectors such as adenylate cyclase or phospholipase C and subsequent generation of second messengers such as cAMP (cyclic adenosine-monophosphate) or calcium. These effects are amplified and transmitted down through a cascade of intracellular events leading eventually to the physiological response of the cell to the stimulus. The enormous diversity of receptors, G-proteins and effectors, together with the widespread distribution of receptors across many tissues, reflects the important role that this family of genes plays in regulating physiological and pathophysiological processes.
The characteristic “motif” of the GPCR family are the 7 distinct hydrophobic regions, each 20 to 30 amino acids in length, generally regarded as forming the transmembrane domains of these integral membrane proteins. Indeed, the alternative name for this family is that of “7TM receptors.” There is little conservation of amino acid sequence across the entire superfamily of receptors, but key sequence motifs can be found within phylogenetically related subfamilies, and these motifs can be used to help classify new members.
Since the first cloning of a GPCR more than a decade ago, over a thousand members of the family have been cloned from a variety of different species. This includes more than 160 distinct sub-types of human receptor for which the natural ligand is known, as well as over 100 human-derived receptor sequences for which the cognate ligand remains to be identified. The sequence motifs exhibited by these “orphan” receptors places them firmly in the GPCR family, but they typically show very low sequence similarity to specific known receptors, generally less than 40%. They are distributed throughout the GPCR phylogenetic tree, and many show better sequence similarity to each other than to known GPCRs, suggesting that they may represent new subfamilies of receptors with distinct, possibly novel, ligands. The majority of orphan receptors have been derived as a result of large-scale DNA sequencing, and as the generation of genomic information continues to increase, so the number of orphan receptors identified in sequence databases continues to increase. There is considerable debate concerning the total number of GPCRs that exist in the human genome, and estimates vary widely from 400 up to 5000. According to the first draft of the entire human genome published as part of the Human Genome Project, there are 616 human GPCRs if only rhodopsin-class, secretin-class, and metabotropic glutamate-class GPCRs are included (J. Craig Venter et al., Table 19 in Science 291: 1304-51).
Indeed, the current human genome sequencing efforts are identifying vast numbers of DNA sequences that may encode receptors, in general, for which corresponding ligands have not yet been identified. In some circumstances, the physiological events in which these orphan receptors are involved are not yet known either, and reagents for elucidating the receptor's physiological function is of importance. In other instances, the receptor is known to play an important physiological role and thereby could provide a means for developing therapeutics for diseases in which these receptors play a role.
The overall strategy for characterizing orphan receptors is often referred to as a “reverse pharmacology” approach to distinguish if from more conventional drug discovery approaches. The conventional approach was historically initiated by the discovery of a biological activity for which the ligand responsible was identified and then used to characterize tissue pharmacology and physiological role. Subsequently, the ligand was used to clone its corresponding receptor for use as a drug target in high-throughput screening. The reverse approach starts with an orphan receptor of unknown function that is used as a “hook” to fish out its ligand. The ligand is then used to explore the biological and pathophysiological role of the receptor. High-throughput screening is initiated on the receptor in parallel with the biological characterization in order to develop antagonists that will help determine the therapeutic value of the receptor.
One of the many great challenges in biology and medicine is to decipher the function of orphan receptors and their mechanism of action. However, traditional efforts to identify ligands for orphan receptors can be inefficient because they involve methodical searches through likely tissue sources to identify the natural ligand for the orphan receptor of interest. There is currently a need to be able to activate a receptor without knowledge of its ligand in order to mimic the effects of ligand binding. Such activated systems can be used to develop functional cell-based and biochemical assays, e.g., for drug screening, as well as to better understand the signal transduction process into which receptor integrates.
Another aspect of receptor-mediated signaling is that constitutively activating mutations have been identified in members of virtually every receptor family. Moreover, such activating mutations have been implicated in a variety of pathological conditions.
To further illustrate, constitutively active G protein-coupled receptors (GPCRs) were first identified in chimeras of the α1- and β2-adrenergic receptors. Ultimately, this effect was mapped to residues at the C-terminal end of the third intracellular loop, and, in particular, the replacement of an alanine at position 293 with any other residue was found to increase the basal activity of the receptor and enhance the affinity for ligand as much as 100 fold. After the identification of naturally occurring constitutively active MC1-Rs and rhodopsin molecules, activating mutations in GPCRs were found to be responsible for a diverse array of inherited as well as somatic genetic disorders including hyperfunctioning thyroid adenomas, autosomal dominant hyperthyroidism, familial precocious male puberty, mettaphyseal chondrodysplasia, familial hypoparathyroidism, and congenital night blindness.
While constitutively activating mutations have been found in virtually all domains of the GPCRs, some mechanistic similarities are commonly found. Many constitutively active receptors demonstrate a higher affinity for agonist and lower EC50 for further activation. In some cases the increased affinity for agonist, but not antagonist, was dramatic, and the correlation between agonist efficacy and increased affinity in the constitutively active mutants led to a proposed modification of the ternary complex model for GPCR activation. The established model holds that agonist binding stabilizes the active conformation (R*G) of the receptor in a complex with G protein while antagonists typically bind equally well to R and R*. Based on the identification and characterization of constitutively active GPCRs, an extended or allosteric ternary complex model was proposed in which receptor, independent of ligand binding, is in equilibrium between an inactive and active conformation. Mutations that constitutively activate receptors are proposed to disrupt internal constraints in the receptors, make the receptors less conformationally constrained, and therefore decrease the energy required to reach the R* state. The model thus explains the increased affinity of agonists for constitutively active receptors, even in the absence of G protein, since constitutive activation results in a higher percentage of receptors in the high-affinity R* state.
Because of the prevalence of constitutively active mutants of GPCRs and other extracellular receptors, there is currently a need to be able to activate a receptor without knowledge of its ligand in order to study the mechanism of action by which activating mutants give rise to disease states.